Abstract Post-traumatic stress disorder (PTSD) is a debilitating disease involving intrusive memories of a traumatic event, which are due in part to an inability to modify responses to stimuli that are no longer threatening?a process known as extinction. The medial prefrontal cortex (mPFC) plays a critical role in extinction by regulating amygdala activity, but the circuit-level mechanisms that support extinction learning are incompletely understood. Converging data indicate that extinction memories are stored in the mPFC through mechanisms that induce synaptic plasticity, new protein synthesis, and potentiated single unit activity. Using two-photon (2P) microscopy, a recent report showed that extinction learning is strongly correlated with spine formation, however, it remains unclear whether and how synaptic remodeling in PFC influences activity in extinction- related circuits, partly because most amygdala-projecting PFC cells reside deep in the medial wall of the PFC, beyond the range of transcranial 2P imaging. Furthermore, the molecular mechanisms that drive extinction- related synapse formation remain unclear. Endocannabinoid (eCB) signaling is a promising candidate. The cannabinoid receptor 1 (CB1R) is the primary eCB receptor in the brain, and deletion of these receptors in mice impairs the acquisition of extinction. eCBs are well positioned to regulate dendritic spine remodeling: they bind post-synaptic CB1Rs, which in turn interact with key regulators of the actin cytoskeleton. Importantly, extinction learning is strongly enhanced in humans with a common SNP in the fatty acid amide hydrolase (FAAH) gene, resulting in increased anandamide levels (one of two eCBs) and enhanced CB1R signaling. Similarly, extinction learning and fronto-amygdala connectivity are also enhanced in human variant FAAH knock-in (KI) mice, which were generated by my co-mentor (Francis Lee) to carry the variant FAAH gene and provide a unique, clinically-relevant tool for investigating plasticity mechanisms underlying extinction memory formation. This project will investigate how postsynaptic dendritic spine formation and eCB-mediated plasticity mechanisms support the encoding of an extinction memory trace within the mPFC. My central hypothesis is that eCB signaling facilitates extinction learning by enhancing postsynaptic dendritic spine formation and driving the emergence of tone-sensitive, multicellular ensembles of amygdala-projecting mPFC cells. I will test this hypothesis at three levels of analysis, leveraging new technologies for optically deleting newly formed synapses in the living brain and for 2P imaging of mPFC through chronically implanted microprisms. Completion of these aims will reveal fundamental insights about the circuit-level mechanisms that support extinction learning and define potentially causal roles for synapse formation and eCB signaling. This project also has direct clinical applications for PTSD as it has the potential to tailor extinction-based treatments for patient populations with the FAAH SNP and open new avenues for treatment development